Development of efficacious strategies for treatment of glioblastoma multiforme (GBM) remains a significant challenge in both pediatric and adult patients. Some improvements, such as treatment with radiation and temozolomide (TMZ), have led to increased survival. However, the prognosis for brain tumor patients remains poor, and is largely due to the ability of these malignancies to acquire chemoresistance by modulation of p53-regulated signaling pathways that control cell survival. Our long-term goal is to develop therapeutic strategies that sensitize drug- and radiation-resistant brain tumors to therapy. In this proposal, we will investigate the efficacy of a novel combination therapy that targets the HDM2/p53 network and DNA repair. Inhibition of HDM2 interactions with key signaling molecules-p53, p73a, and HIF1a-by the small molecule inhibitor, nutlin3, can modulate their downstream effector function. Depending on the cell type studied, exposure to the HDM2 antagonist can lead to cell cycle arrest, senescence, apoptosis, decreased migration, and attenuation of VEGF production. To what extent TMZ and radiation in combination with nutlin3 can modulate these critical intracellular targets has not been studied. Our data indicate that nutlin3 can significantly potentiate TMZ- and radiation-mediated cytotoxicity in glioblastoma cells in vitro. In addition, nutlin3 also enhanced TMZ-mediated glioblastoma cell kill in an ectopic xenograft model. Our overall objective is to develop efficacious treatment strategies that kill brain tumor cells but not normal cells. For drug efficacy studies, ectopic and orthotopic glioblastomas will be established in NOD/SCID/IL2Rnull mice. A panel of established glioblastoma cell lines and early passaged glioblastoma primary cultures that differ in EGFR gene amplification, p53 status (wild-type or mutant), HDM2 status, MGMT expression, and sensitivities to TMZ and irradiation will be utilized. Real-time bioluminescence imaging will be utilized to serially monitor glioblastoma progression over time. Our central hypothesis is that nutlin3 potentiates the TMZ- and/or radiation-induced DNA damage response by perturbing HDM2-mediated regulation of key signaling molecules, and leads to increased glioblastoma cell death in vivo. To test this hypothesis, the following specific aims are proposed: 1) Develop therapeutic regimens and validate intracellular target modulation mediated by inhibition of HDM2-protein interactions during exposure to DNA-damaging agents 2) Assess in vivo the outcome of modulating HDM2-dependent signaling to increase therapeutic efficacy of TMZ- and/or radiation- mediated DNA damage. 3) Employ intracranial GBM xenograft models in combination with serial real- time bioluminescence imaging to monitor therapeutic impact of modulating HDM2-dependent signaling in combination with TMZ- and/or radiation-mediated DNA damage. The treatment strategies investigated here will use clinically relevant in vivo models and novel multi-targeting approaches and have the potential to improve treatment efficacy and quality of life for patients with GBM.